Provision of water by halite deliquescence for …...Provision of water by halite deliquescence for...

Provision of water by halite deliquescencefor Nostoc commune biofilms under Marsrelevant surface conditions

Jochen Jänchen1, Nina Feyh2, Ulrich Szewzyk2 and Jean-Pierre P. de Vera31TH Wildau (Technical University of Applied Sciences), Hochschulring 1, 15745 Wildau, Germany e-mail: [email protected] Berlin, Institute of Environmental Technology, Environmental Microbiology, Ernst-Reuter-Platz 1, Berlin, 10587Berlin, Germany3DLR Institute of Planetary Research, Rutherfordstr. 2, D-12489 Berlin, Germany

Abstract: Motivated by findings of new mineral related water sources for organisms under extremely dryconditions on Earth we studied in an interdisciplinary approach the water sorption behaviour of halite, soilcomponent and terrestrial Nostoc commune biofilm under Mars relevant environmental conditions.Physicochemical methods served for the determination of water sorption equilibrium data and survival ofheterotrophic bacteria in biofilm samples with different water contents was assured by recultivation.Deliquescence of halite provides liquid water at temperatures

there is a lack of knowledge about the heterotrophic micro-biota of terrestrial N. commune biofilms.If a biofilm formed by a primary producer would be pro-

vided with water by deliquescence of halite under Martianconditions, it could serve as a habitat for other bacteria aswell. Thus we put an emphasis on the identification of all pro-karyotes in the biofilm samples and the viability of associatedmicroorganisms. This was done by a test to ascertain a possibleinfluence of desiccation, water content and salinity on reculti-vation. In addition to halite and biofilm we includedmontmor-illonite in our study, as it has been identified in theMartian soil(Poulet et al. 2005; Vaniman et al. 2014) and may form associ-ates with bacteria being beneficial for their metabolism andsurvival (reviewed by Marshall 1975 and England et al.1993) as well as being a source of water supply.Palacio et al. (2014) discusses even the crystallization water of

otherminerals like gypsumas a relevantwater source for plants inan extremely dry environment. Because huge deposits of sulphatehydrates are well known to occur on theMartian surface (Bibringet al. 2005; Gendrin et al. 2005) crystallization water may poten-tially be another water source for microorganisms on Mars.Our interdisciplinary study of a physicochemical and micro-

biological approach to an astrobiological issue should help toevaluate cryobrines on Mars as potential habitats and therebygain knowledge in order to support missions such as MarsScience Laboratory (MSL) and ExoMars/MicrOmega to rec-ognize interesting habitable Martian sites. Very recentlyMartín-Torres et al. (2015) reported about evidence of night-time transient liquid brines (perchlorate based) in the upper-most subsurface of Gale crater in evaluating data of theCuriosity rover (MSL). The science team also found changesin the hydration state night/day of salts consistent with an ac-tive exchange of humidity between atmosphere and uppermostsoil surface.In this sense extremotolerant organisms are under current in-

vestigation with respect to survival of space and Mars condi-tions to gain knowledge about the boundaries of life beyondEarth. Therefore the extremotolerant organisms such as li-chens and cyanobacteria are part of the current EXPOSE-R2Biology andMars experiment (BIOMEX) on the internationalspace station (Baqué et al. 2013; Böttger et al. 2012; de Veraet al. 2012, 2014; Billi et al. 2013; Meeßen et al. 2013a, b).Finally, our study may contribute to a better understandingof the speciation of adsorbed H2O, hydrated and hydroxylatedphase on the Martian surface (Jouglet et al. 2007; Vanimanet al. 2014).

Materials and methods

Materials

Halite, a palm sized piece, from the hyper-arid core of theAtacama Desert (Yungay region, Chile, donor AlfonsoF. Davila, Davila et al. 2008) have been employed in thisstudy. For comparison, in particular for the hydration proper-ties, very pure NaCl (Merck, 99.99%) and the smectiteCa-montmorillonite (STx, Gonzales County, Texas; source

Clay Minerals Repository, 101 Geological Science Bldg.,Columbia, MO 65211, USA) have been included in this study.All experiments concerningN. commune were performed on

natural colonies, collected dry from a concrete bridge deck inthe national park ‘Unteres Odertal’ (53°8′2″N, 14°22′24″E;Brandenburg, Germany). For identification of associated bac-teria, two different biofilm samples were investigated in add-ition: another sample, that was collected wet at the bridgesite in the national park described above and dried for 20d ina sterile petri dish over silica gel prior to analysis and dry col-onies from a flat rooftop (50°23′45″N, 9°1′32″E; Hesse,Germany). The dry colony material has been stored for 5(national park) or 4 (rooftop) years in sterile glass flasks orpolypropylene tubes at room temperature in the dark.

Methods

Sorption and thermal methods

The hydration/dehydration properties of Atacama halite, pureNaCl, montmorillonite and N. commune were investigated bymeans of isotherm measurements and thermoanalysis such asthermogravimetry (TG), differential thermogravimetry (DTG)anddifferential thermoanalysis (DTA). Sorption isothermsweremeasured gravimetrically from 256 to 293 K with a McBain–Bakr quartz spring balance (McBain & Bakr 1926) equippedwith threeMKS Instruments Inc. (MKS)Baratron pressure sen-sors covering a range of 10−5–103 mbar. The sensitivity of thequartz spring was 4 mg mm−1. The extension of the springwasmeasurablewith a resolution of 0.01 mmgiving a resolutionof 0.04 mg for the quartz spring. In terms of ‘g water/g dry sor-bent’ this results ina resolutionof 0.0004 g g−1 for applicationof100 mg sample or in the case of 400 mg to 0.0001 g g−1.TG, DTG and DTA measurements were performed on a

Netzsch STA 409 apparatus with a heating rate of 10 Kmin−1 up to 600 or 900 K and a purge gas stream (nitrogen)of 70 ml min−1. Prior to the experiments all samples hadbeen stored in controlled atmosphere (evacuated desiccator):N. commune and montmorillonite at p/ps H2O = 0.79 (relativehumidity (RH) = 79%) and halite or NaCl at p/ps H2O= 0.60(RH= 60%) for several days.Scanning electron microscopy (SEM, JOEL JSM640 and

ZEISS Gemini Ultra Plus) combined with energy dispersiveX-ray analysis (EDX) was applied to characterize the morph-ology and chemistry of the salt samples.Before each sorption experiment, about 150 mg of sample

(or 350 mg in case of the salts) had been degassed in high vac-uum (p< 10−5 mbar) at 293 K overnight (N. commune and thesalts for an extra run) as well as at 383 K (montmorillonite) and413 K (salts) for several hours. The degassing temperatureswere limited to individual adapted low value to prevent pos-sible modification of the mineral- and biofilm-samples.

Recultivation

N. commune samples used in the survival experiment(5-year-old, Odertal) had been equilibrated in a desiccatorover silica gel (RH= 30–40%). Samples were then incubatedunder the following conditions: in a desiccator over sterile

108 Jochen Jänchen et al.

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saturated NaCl (RH= 75%) solution (19d at 20.2 ± 0.7 °C) orcompletely covered in sterile saturated NaCl solution (for 14dat 20.2 ± 0.8 °C). Three silica gel dried replicate samples servedas a reference and five samples per treatment were analysed.One autoclaved (134 °C for 30 min) sample per treatmentserved as sterility control.Survival of associated heterotrophic bacteria was determined

by spiral plating (IUL EddyJet spiral plater) of homogenizedsample material on low-nutrient Reasoner’s 2A (R2A) agarplates. Samples were homogenized in sterile 1× phosphatebuffered saline (PBS) for 2 min at 6000 rpm with an IKAULTRA-TURRAX Tube Drive homogenizer. Three sub-samples of each homogenizate were serially diluted with 1×PBS. After incubation at 20.3 ± 0.7 °C for 7d, the colonieswere counted.Statistical analysis and plotting of heterotrophic plate counts

was performed using the R statistics software environment (RCore Team 2013). A non-parametric test (Kruskal–Wallis) wasused to check for significant variation of results among differ-ent treatment groups.

Microscopy and confocal laser scanning microscopy(CLSM)

Phase-contrast light microscopy was performed on dried biofilmpieces mounted with Citifluor AF2 (Citifluor Ltd.) with a ZeissAxioplan microscope equipped with a 63× Plan-Apochromatobjective. For CLSM, biofilms were prepared as follows: sam-ples were covered in sterile ultrapure water (deionized (DI)water filtered with Satorius Sartopore 2, 0.2 μm) and allowedto swell for 10 min. After flattening the sample by squeezing be-tween two object slides, 10 μl of 1 : 1000 diluted SYBR Green Ifluorescent dye (Life Technologies) was applied followed by in-cubation for 5 min in the dark and twofold rinsingwith ultrapurewater. A Leica TCS SP5II CLSM equipped with an HCX PLAPO CS 100× objective was used to acquire image data (excita-tion wavelengths: 458/496 nm). CLSM data were processed withthe software distribution Fiji (Schindelin et al. 2012).

Uptake of saturated NaCl and MgCl2 solutions

Silica gel dry biofilmmaterial (four replicates per treatment) wasweighed before and after incubation in brine for 24 h at 22.5 ±0.2 °C. One parallel of samples was incubated in DI water.Samples were filter centrifuged in tube filters (Corning CostarSpin-X with 0.2 μm cellulose acetate membrane) at 9500 g for1 min to remove loosely associated brine or water. After weight-ing the samples their content of soluble was determined as fol-lows: homogenization in DI water as described above for therecultivation experiment and filtration through glass fibre filters(MACHEREY-NAGEL MN 85/70). After evaporating the li-quid in a drying oven at 110 °C, the remaining solid phases wereweighed. For calculation of salt contents, the average solidphase mass of the DI water parallel was subtracted from the va-lues obtained from the salt containing samples.

16S rDNA clone libraries

Accompanying bacteria were extracted as follows: biofilm ma-terial (100–500 mg) was incubated in 10 ml sterile 1×Phosphate Buffered Saline (PBS) for 1 h and homogenizedand filtered as described above for the determination of liquiduptake. Cells were pelleted by centrifugation at 4000 g for 10min and washed once with 1× Phosphate Buffered Saline(PBS). Gene matrix Soil DNA Purification Kit (EURx) wasused for DNA extraction. Cloning (TOPcloner™ TA kit,Enzynomics) and sequencing with primer M13F was carriedout by Macrogen Inc., South Korea.Vector sequences including primer region and low-quality

ends were trimmed manually by means of DNA Baser(DNA Baser Sequence Assembler v4.x (2014),HeracleBioSoft SRL, www.DnaBaser.com). Clone librarieswere checked for chimeric sequences using DECIPHER(Wright et al. 2012) and suspicious sequences were removed.Ribosomal database project Classifier Version 2.8 was usedfor classification of clone sequences with an assignment confi-dence cut-off of 80% according to Bergey’s Taxonomic Outlineof the Prokaryotes (Wang et al. 2007).

Fig. 1. SEM images of Atacama halite (left) with impurity CaSO4·2H2O (mark, identified by EDX) and NaCl, purity 99.99% (right).

Provision of water by halite deliquescence for Nostoc commune biofilms 109


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Results and discussion

Characterization of the samples

Halite and sodium chloride consist of nonporous crystals dif-ferent to the layered structure of the smectite montmoreillonite.Sorption of water vapour can occur in the inner pore systemand/or on the outer surface of the particles. Montmorilloniteis able to sorb bigger amounts of water (see later) in agreementwith the ability of the polar water molecule to penetrate intothe interlayer spaces. The salt crystals offer the outer surfacefor sorption only which is a few orders of magnitude lesssurface area for sorption of water molecules as for the smectite.The SEM images (Fig. 1) give information about the morph-ology and particle size of the samples. Halite (left) shows par-ticles of irregular shape and the pure NaCl indicate moreregular cube-shaped particles as expected. The particle sizedistribution for both samples seems to be roughly the samewith 10–100 μm generating a surface area of about 0.06 m2

g−1 much less compared with 200 m2 g−1 of the smectite(related to water).The EDX analysis of NaCl shows exclusively Na and Cl.

Halite is almost pure NaCl but with some impurities of gypsum(CaSO4) and an aluminosilicateas identified by EDX (map-ping). Themarked particle on the image (left part of Fig. 1) con-sists of the elements Ca, S, O forming most probably gypsum.N. commune biofilms are shown in Fig. 2. Dry Biofilms are

rather compact and brittle and swell to thin gelatinous layersafter being wetted. Filaments of spherical cells typical forNostoc species can be seen in wet biofilms. Single cells andmicro colonies of smaller bacteria are located on the irregularlyshaped EPS surface, which can be observed by light micros-copy as well as by CLSM of fluorescent stained biofilms(Fig. 3). None of these cells are visible within the EPS whereascavities are inhabited. These accompanying bacteria mightregularly colonize surfaces of N. commune biofilms to take ad-vantage of the protective function of their EPS against desicca-tion or UV radiation. A provision of nutrients excreted bycyanobacterial cells is also conceivable.

Water sorption properties of the materials at equilibrium

Thermoanalysis gives first of all a quick overview of the waterrelease and at further rising temperatures about the dehydrox-ylation and decomposition of the samples. Figure 4 sum-marizes the TG/DTG/DTA data for N. commune and Fig. 5the TG/DTG data for halite and NaCl. The initial mass loss(desorption of H2O) ranges from 12 wt.% for N. commune totiny amounts of less than 0.3 wt.% for halite. Halite andNaCl (cf. Fig. 5) do not form hydrates above 273 K and there-fore does not decompose such as other hydrate forming saltsusually do. In particular the pure NaCl does not show anymass loss up to 600 K. Natural halite containing traces of gyp-sum behaves differently. Gypsum forms a dihydrate upon con-tact with water vapour. Halite in Fig. 5 shows mass loss ofabout 0.15 wt.% at 403 K very close to the temperature typicalfor mass losses of gypsum because of release of its crystalliza-tion water (Paulik et al. 1992).N. commune decomposes in two steps (Fig. 4) starting with

weakly bonded water (endothermic step at 345 K) followed bya considerable mass loss between 500 and 600 K due to furtherdehydration and dehydroxylation of the organic polymers and,finally, anaerobe decomposition of the entire material. Thisprocess is partially exothermic (cf. DTA) different to the heatconsuming dehydration at the beginning of the TG and DTAcurve. The residual material looks black like carbon what canbe explained by the occurrence of pyrolysis.Because our paper focuses until now on the physical inter-

action of water vapour with the biofilm, the first TG-step isof special attention (the reversibly bonded water at T< 400K, Fig. 4). Further, the question arises: Is there any water up-take of the halite under defined humid conditions and low tem-peratures? Isotherm measurements can give that informationin terms of equilibrium data of physically bonded water.Fig. 6 shows the H2O sorption isotherms of halite at differentlow Mars relevant temperatures. Figure 7 compares the iso-therms of halite and NaCl at 293 K in more detail. As can beseen the Atacama halite and NaCl do not sorb much water

Fig. 2. N. commune: dry biofilm (A); stereo microscope micrograph of wet surface with visible N. commune cell filaments (B); phase contrastmicrograph of bacterial microcolony (black arrow) on EPS surface (white arrow) (C).




below the deliquescence RH (DRH) = 75%. Above this valueboth salts start gaining weight due to forming a wet skin of sa-turated solution on the crystal’s surface (Davila et al. 2010;Hansen-Goos et al. 2014). Deliquescence continues at constantRH converting the solid salt surface step by step into a concen-trated salt solution. Interestingly, the DRH depends somewhaton temperature in our experiments.A closer look to the water sorption below the deliquescence

point is given for 293 K in Fig. 7 (note the logarithmic scale ofthe ordinate). Sorbed amounts of 0.0002–0.002 g g−1 forRH< 75% could be detected. Despite being close to the reso-lution of the method (see above) a tendency of higher sorbedamounts for halite degassed at elevated temperature is likely.

Owing to the gypsum impurity of the halite the crystallizationwater of gypsum which had been removed at the elevated tem-perature is sorbed afterwards as extra amount by hydration ofgypsum. In the case of degassing at room temperature the hal-ite behaves like the pureNaCl taking some less water. This verysmall amount of water is due to the formation of pre-deliquescence layers on the crystal surfaces as reported byBruzewicz et al. (2011) and Hansen-Goos et al. (2014). Layerheights of some nm were calculated by Hansen-Goos in ac-cordance with our findings taking into account the surfacearea estimated for the crystals in halite or the pure NaCl.The hydration and dehydration isotherms of N. commune

are displayed in Fig. 8 for temperatures of 257–293 K. The

Fig. 3. CLSM scans of N. commune biofilm shortly after wetting: top views at different depths and corresponding cross-sections (depth andposition of sections are indicated by cross-hairs); SybrGreen I stained small bacterial cells are displayed in green and N. commune cells in red(chlorophyll autofluorescence), DNA-rich parts of cyanobacterial cells appear yellow due to mixing of both colours.




sorbed amount of about 0.17 g water g−1 vacuum dry sampleat RH= 80% corresponds to the first TG-step (weakly bondedH2O below 400 K) ofN. commune in Fig. 4. A detailed study ofthis ‘reversible’water sorption was thus carried out by isothermmeasurements. The amount of adsorbed water varies with therelative vapour pressure (0.001–0.9, corresponding to 0.1–90%RH in Fig. 8) and amounts to 0.01–0.25 g g−1 (correspondingto 1–25%). Thus about 25 wt.% water represents the somewhatless-strongly bonded water of the much higher total quantitybearing by the biofilm.Striking is the pronounced hysteresis between hydration and

dehydration not observed for the lichen Xanthoria elegans orLeptothrix biofilms (cf. Jänchen et al. 2014). This may indicatea superior ability of N. commune to store water if compared

with lichens and Leptothrix biofilms. Cyanobacterial photo-bionts as part of a lichen symbiosis are known to have an ele-vated water storage capacity and can contain more water in thesaturated state than algal photobionts due to the formation oflarger layers and the production of EPS (Gauslaa & Coxson2011). This has been explained by the observation, that liche-nized algal photobionts can become photosynthetically activeby uptake of humidity from the air, whereas lichens with acyanobacterial photobiont and terrestrialN. commune coloniesdepend on the direct uptake of liquid water (e.g. from rain or bycondensation of water vapour) to perform photosynthesis(Lange et al. 1986, 1993).N. commune takes advantage of the ability to excrete large

amounts of EPS and thus to save water in the biofilm for

Fig. 4. TG (solid green line), DTG (dashed-dotted blue line) and DTA (dotted red line) profiles for N. commune.

Fig. 5. TG (solid line) and DTG (dashed-dotted line) of halite (green) and NaCl, Merck 99,99% (solid red line, top) stored at RH= 60% prior tothe experiments, note the TG scaling compared with Fig. 4




longer periods at lower RH. High moisture absorption andretention capacities have been reported as well for isolatedpolysaccharides ofN. commune (Li et al. 2011). As a result, des-iccation is slowed down and thus the period, when photosyn-thesis is possible is extended (Gauslaa et al. 2012).Figure 9 gives a comparison of the hydration and dehydra-

tion characteristics of N. commune with montmorillonite andhalite as function of the RH. This comparison holds well forT≪ 293 K and shows N. commune as much more hydrophilicthan halite and similarly hydrophilic as the smectites. Thus hal-ite can provide N. commune with liquid water because of deli-quescence at the DRH= 75–80% even below 273 K.Application of the Dubinin equation on the isotherm data ofN. commune (Fig. 6), as shown in (Jänchen et al. 2006) forthe smectite, provides the data in a water amount/temperatureplot at a certain water vapour pressure.Thus detailed information about the physicochemical inter-

action of halite, montmorillonite and N. commune with watervapour and the deliquescence of halite forming a saturated so-lution on its surface has been obtained and documented in

Fig. 9. However, it is also important to have informationabout the desiccation and saline tolerance of N. communeand the associated heterotrophic bacteria as part of the biofilmin contact with liquid phases. The following sections draw at-tention to this issue.

Recultivation of heterotrophic bacteria from N. communesamples

Heterotrophic plate counts for different treatments of biofilmsare shown inFig. 10. Colony-FormingUnit (CFU) numbers ob-tained from the silica gel dry controls, which have been in a drystate for at least 5 years were in a range of 6.2 × 106–2.3 × 107

g−1 dry weight, which is comparable with experimental dataof various soil samples yielded under similar cultivation condi-tions (Iivanainen et al. 1997; Adesina et al. 2007; Desai et al.2009). Water uptake at RH= 75% (14.13 ± 0.76 wt.%) didnot lead to a significant change in CFU number comparedwith the silica gel dry controls. A 24-fold decrease of CFUwas observed for samples incubated in NaCl brine. All sterilitycontrols showed no growth.

Fig. 6. Water isotherms of Atacama halite upon outgassing at 413 Kat different temperatures: 256 K (squares), 273 K (triangles); filledsymbols and dashed line denote dehydration.

Fig. 7. Water isotherms of halite (triangles, circles) and NaCl(diamonds, squares) at 293 K upon outgassing at room temperature inhigh vacuum overnight (triangles, diamonds) or outgassing at 413 K inhigh vacuum (circles, squares). Filled symbols and dashed lines denotedehydration. Note the extended view of the low sorbed amounts(RH< 70%) by logarithmic scaling.

Fig. 8. Water sorption isotherms (water vapour uptake and release atequilibrium) forN. commune at 257 K (triangles), 273 K (squares) and293 K (first run diamonds, second run squares), filled symbols anddashed lines denote dehydration.

Fig. 9. Comparison of water isotherms at 273 K for montmorillonite(squares), N. commune (circles) and halite (triangles); filled symbolsand dashed lines denote dehydration.




Our results suggest a high desiccation tolerance of the asso-ciated heterotrophs. This could be explained by an adaptationof the biofilm’s microbiota to the lifestyle of N. commune. Asalready mentioned in section ‘Water sorption properties of thematerials at equilibrium’ the cyanobacterium needs to be incontact with liquid water to perform photosynthesis and thushas to be able to endure long periods of desiccation betweenevents of rain or condensation of water (Lange et al. 1986,1993; Gauslaa et al. 2012). This would also explain the lackingbenefit from uptake of small amounts of water for recultivabil-ity, since it would be unfavourable for the accompanying

Fig. 10. Heterotrophic plate counts (logarithmic scale) per g silica gel dry sample for three different treatments: storage over silica gel untilequilibration, at RH= 75% or in saturated NaCl solution.

Table 1. Liquid uptake byN. commune: weight percentages ofliquids (taken up by whole biofilms), solubles/remnants (of wethomogenized biofilms after filtration) and calculated mass frac-tions of salts in the brine (values are means ± standarddeviations)

SolutionUptake of water/brine (wt.%)

Solubles(wt.%)

Calculated mass frac-tions of salts (wt. %)

DI water 911.5 ± 222.6 16.5 ± 5.3 –MgCl2 309.8 ± 115.6 85.5 ± 4.7 31.1 ± 9.8NaCl 367.3 ± 54.3 98.6 ± 34.9 25.4 ± 7.6

Fig. 11. Comparison of clone libraries from long-time desiccatedsamples WH2 and N1 together with the one from short-timedesiccated sample OT1: percentage of clone sequences assigned todifferent taxa and unclassified groups.




microbiota to become metabolically active under circum-stances, where the biofilm host is not close to leaving dor-mancy. Survival of long periods in a desiccated state by theheterotrophs could also be enhanced by a protective functionof the cyanobacterial EPS. Since the EPS surface has an irregu-lar shape (especially, when the biofilm is dry, see Figs. 2 and 3),small bacterial cells are enclosed in cavities. Beside a protectionagainst mechanical stress, this sheltered position can also helpto avoid UV damage.The brine treatment showed that only a fraction of 4% of the

heterotrophs can grow under highly saline conditions. This is inagreement with the moderate tolerance of the biofilm forminghost to NaCl resulting in an inhibition of photosynthesis at a

concentration of 0.2 M or 30 g kg−1 (0.51 M) (Sakamotoet al. 2009; Sand-Jensen & Jespersen 2012).

Uptake of water, NaCl and MgCl2 brines by N. communebiofilm

The uptake of DI water or saturated NaCl/MgCl2 solutions bydry biofilms within 24 h is listed in Table 1. While an amountof around 900% water was taken up, the salt solutions contrib-uted to approximately a third of the final sample weight. Themeasured salt contents in the brines taken up were near the va-lues of saturated solutions at 20 °C for NaCl (26.4 wt.%) andMgCl2 (35.3 wt.%).

Table 2. Clone sequences classified on genus level and the share of each phylum/class to the total clone sequence number in the samplelibrary

Clone sequences

Phylum/class Genus WH2 (long time) N1 (long time) OT1 (long time)

Actinobacteria Actinoplanes 0 0 0∑/Percentage share 0/0% 0/0% 1/2.2%Alphaproteobacteria Aurantimonas 0 0 1

Brevundimonas 1 4 0Caulobacter 2 1 1Unclassified Caulobacteraceae 0 1 0Devosia 0 0 1Hyphomonas 0 1 0Unclassified Hyphomonadaceae 0 0 1Mesorhizobium 1 0 0Methylobacterium 1 1 5Microvirga 1 0 0Novosphingobium 1 0 0Phenylobacterium 1 1 0Rhizobium 3 0 0Unclassified Rhizobiales 0 2 0Rhizomicrobium 1 0 0Rubritepida 0 0 1Sphingobium 0 1 0Sphingomonas 17 18 4Unclassified Sphingomonadaceae 2 7 0Unclassified Sphingomonadales 0 1 0

∑/Percentage share 31/72.1% 38/92.7% 14/31.1%Bacteriodetes Adhaeribacter 0 2 0

Hymenobacter 0 0 8Mucilaginibacter 0 1 2Pedobacter 2 0 0

∑/Percentage share 2/4.7% 3/7.3% 10/22.2Betaproteobacteria Limnobacter 1 0 0

Roseateles 1 0 0Shinella 1 0 0

∑/Percentage share 3/7.0% 0/0% 0/0%Chloroflexi Sphaerobacter 0 0 1∑/Percentage share 0/0% 0/0% 1/2.2%Cyanobacteria Chlorophyta 0 0 1

Gpl 0 0 16∑/Percentage share 0/0% 0/0% 17/37.8%Gammaproteobacteria Enterobacter 1 0 0

Unclassified Enterobacteriaceae 4 0 0Lysobacter 0 0 1Pseudomonas 2 0 0Unclassified Gammaproteobacteria 0 0 1

∑/Percentage share 7/16.3% 0/0% 2/4.4%




Asmentioned earlier,N. commune has been found to be onlymoderately salt tolerant and thus, the cyanobacterial colonieswould not be viable in such a brine-soaked state. Nevertheless,it has been shown, that a community consisting of cyanobac-teria and heterotrophs can be supported through exclusivewater uptake from a brine formed by deliquescence on sur-rounding halite (Wierzchos et al. 2006; de los Ríos et al.2010; Davila et al. 2013; Robinson et al. 2015).

16S rDNA cloning

Affiliations of clone sequences to different phyla and classesare shown in Fig. 11 and Table 2. High proportions of se-quences were assigned to the Alphaproteobacteria group(31.1–92.7%), to which Sphingomonadaceae constituted themajor part (8.9–61.0%). This was more obvious for the long-time desiccated samples, indicating a high desiccation toler-ance of both groups. Sequences of N. commune (assigned togenus GpI, see Table 2) were only present in the library ofthe short-time desiccated sample OT1. This result might becaused by cyanobacterial cells of long-time desiccated biofilmsbeing less prone to damage during homogenization in thecourse of cell extraction.

Implications of the Martian surface conditions on habitability

A decade ago it was believed that the thin Martian atmosphere(about 6 mbar) with its very low water vapour pressure (about0.001 mbar) cannot offer a liquid phase particularly in warmerequatorial latitudes serving as water source for possible bio-logical activities. But the Martian soil should offer at leastfor some hours in a diurnal circle a liquid water phase (by con-densation or hydration of minerals) for the organisms to be-come viable in the proposed biofilms being in contact withthe ‘wet’ soil components. The biofilmmay extend the presenceof water because of its storage capability but the sourcemust bethe atmosphere. Möhlmann suggested three different sourcesof liquid water feed by the atmosphere in equatorial latitudesat the Martian surface: adsorption water on mineral surfaces,water from temporary melting processes in upper sub-surfaceparts of snow/icepacks and cryobrines formed by deliquescence

of salts (Möhlmann 2008, 2010a, b). The formation of so-called cryobrines on salts such as halite is one of the nomineesto provide a liquid phase under close toMartian environmentalconditions for possible biological activity beside geological andchemical processes (Möhlmann 2011).Perchlorate measured at NASA’s Phoenix Lander site and

the formation of possibly liquid perchlorate at the lander’sstud, as discussed by Renno et al. (2009a, b) and Chevrieret al. (2009) as well as the already mentioned results ofCuriosity at Gale crater (Martín-Torres et al. 2015) are in prin-ciple other examples for providing liquid phases on presentMars. Even though a strongly oxidizing perchlorate solutionmight be poisonous for many organisms members of the pro-teobacteria have been found to be able to reduce perchlorate indiluted solution (Coates et al. 1999).Brines formed on halite would be probably more life sup-

porting and a suitable source for water as found for cyanobac-teria in the Atacama Desert. But, as observed in the AtacamaDesert, a potential source of water should stay at least for sometime in a diurnal circle liquid at Martian surface conditions inmid- and low-latitudes. Fig. 12 gives information about the sta-bility of the NaCl brine at the Martian surface for equatoriallatitudes. The straight (red) line in Fig. 12 characterizes thepartial pressure of water vapour in theMartian atmosphere be-tween 230 and 273 K. The progression of the effective vapourpressure of NaCl brine with temperature is documented by theconcave line. The calculated pressure values are lower thanthe equilibrium data because the stability of the brine is betterdue to the presence of the CO2 atmosphere. Hence, approxi-mately 6 mbar (0.06 Pa) CO2 of the Martian atmosphere re-duces the vaporization rate in the diurnal circle (dayvaporization/night condensation and deliquescence) signifi-cantly (based on Taylor et al. 2006). Accordingly, NaClbrine should ‘survive’ below 265 K to sustain biological pro-cesses of halotolerant cyanobacterial biofilm communities.According to our findings a hypothetical Mars bacterial

community might be likely to succeed as life in microhabitatsunder present Martian surface conditions. This communitycould consist of a halotolerant primary producer, which hadto be able, to gain enough energy to produce a large, hydro-philic EPS, similar to N. commune. As shown in section‘Uptake of water, NaCl andMgCl2 brines byN. commune bio-film’, such an EPS is able to take up about 300% of saturatedNaCl brine in direct contact to the liquid, which is then heldback when the conditions get drier due to the water storingproperties of the biofilm matrix. This reservoir can then beused by the primary producer as well as by likewise halotoler-ant heterotrophs inhabiting the biofilm as a protective niche.Clays, having similarly hydrophilic properties as N. commune(see Fig. 9), could add to enhanced water storage as a compo-nent of soil in contact to the biofilm-communities.

Conclusions- Deliquescence of halite at low temperatures (

- The stability of the NaCl cryobrine (concentrated halitesolution) on the Martian surface at equatorial latitudesis better than equilibrium data suggest because of thepresence of the CO2 atmosphere reducing the vaporiza-tion rate (day/night) significantly.

- Biofilms protecting microorganisms on Earth againstdesiccation show a rather high hydrophilic charactercompared with smectites. The biofilm of N. communetends to be able to store water because of the hydrophilicEPS, documented by the hysteresis between the hydra-tion and dehydration branch of the isotherms.

- The water content of the soil components and bio-materials changes with its nature and depends stronglyon temperature and partial pressure. Knowing the localhumidity of a planet’s atmosphere the water content ofthese materials can be determined. Supply of this datamay support the evaluation of spectroscopic resultsfrom orbit or rovers (MSL, ExoMars/MicrOmega).

- A high desiccation tolerance of heterotrophic bacteriaassociated toN. commune was emphasized by recultiva-tion tests. The protective function of a large EPS againstdesiccation would be a benefit as well in a water-limitedenvironment like Mars.

- The heterotrophic microbiota of the investigatedN. com-mune biofilms is dominated by Alphaproteobacteria andmight be adapted to their host’s long-time desiccation en-during lifestyle. Water uptake from saturated NaCl solu-tions (either by uptake of brine or humidity) does notenhance recultivability of the heterotrophs. Part of themicrobiota is far more tolerant to highly saline condi-tions, than the cyanobacterial host.

- Summarizing it can be stated that the current Martiansurface conditions at equatorial latitudes might offerat least temporarily niches with habitable conditionsfor halotolerant organisms. Habitable means the pres-ence of liquid water as brine at surface temperatures ofabout 255–265 K and RH< 80%. A N. commune-typebiofilm together with montmorillonite could in thiscase serve as a habitat for heterotrophic bacteria by up-take and retention of briny water.

Acknowledgements

We thank Alfonso F. Davila for supply of the Atacamahalite sample and Dirk Möhlmann (DLR, Berlin) forhis contribution to section ‘Implications of the Martiansurface conditions on habitability’. This research was sup-ported by the Helmholtz Association through the researchalliance ‘Planetary Evolution and Life’ as well as fundedby a grant of the BMBF (50WB1151) and is part of thecurrent BIOMEX mission (ESA call, 2009, Ref.-No.ILSRA-2009-0834).

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Provision of water by halite deliquescence for Nostoc commune biofilms under Mars relevant surface conditionsAbstractIntroductionMaterials and methodsMaterialsMethodsSorption and thermal methodsRecultivationMicroscopy and confocal laser scanning microscopy (CLSM)Uptake of saturated NaCl and MgCl2 solutions16S rDNA clone libraries

Results and discussionCharacterization of the samplesWater sorption properties of the materials at equilibriumRecultivation of heterotrophic bacteria from N. commune samplesUptake of water, NaCl and MgCl2 brines by N. commune biofilm16S rDNA cloningImplications of the Martian surface conditions on habitability

ConclusionsAcknowledgementsReferences

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